Method for producing citric acid

ABSTRACT

METHOD FOR PRODUCING CITRIC ACID WHICH INVOLVES INOCULATING A CITRIC ACID-ACCUMULATING YEAST BELONGING TO THE GENUS CANDIDA, CAPABLE OF UTILIZING HYDROCARBONS AND INCAPALE OF UTILIZING CITRIC ACID IN AN AQUEOUS CULTURE MEDIUM CONTAINING, AS THE MAIN CARBON SOURCE, AT LEAST ONE NORMAL PARAFFIN CONTAINING 9 TO 20 CARBONS IN THE MOLECULE; INCUBATING THE CULTURE AT A PH OF FROM ABOUT 2 TO 7 UNTIL CITRIC ACID IS SUBSTANTIALLY ACCUMULATED IN THE CULTURE BROTH; AND RECOVERING THE THUS ACCUMULATED CITRIC ACID THEREFROM. THE ADVANTAGES OF THE AFOREMENTIONED METHOD IS THAT THE PRODUCTION OF OTHER ASOCIATED ORGANIC ACIDS IS SUPPRESSED, WHICH RESULTS IN AN INCREASED YIELD OF THE OBJECTIVE CITRIC ACID; THE FERMENTATION TERMINATES IN A RELATIVELY SHORT TIME AND FEMENTATION CAN BE CARRIED OUT WITHOUT FEAR OF OF OVERFERMENTATION SINCE THE CITRIC ACID IS NOT CONSUMED BY THE ORGANISUM.

United States Patent Oflice 3,689,359 Patented Sept. 5, 1972 Int. Cl. c'rzd 1/04 US. Cl. 195-48 R 8 Claims ABSTRACT OF THE DISCLOSURE Method for producing citric acid which involves inoculating a citric acid-accumulating yeast belonging to the genus Candida, capable of utilizing hydrocarbons and incapable of utilizing citric acid in an aqueous culture medium containing, as the main carbon source, at least one normal paraffin containing 9 to 20 carbons in the molecule; incubating the culture at a pH of from about 2 to 7 until citric acid is substantially accumulated in the culture broth; and recovering the thus accumulated citric acid therefrom. The advantages of the aforementioned method is that the production of other associated organic acids is suppressed, which results in an increased yield of the objective citric acid; the fermentation terminates in a relatively short time and fermentation can be carried out without fear of overfermentation since the citric acid is not consumed by the organism.

This invention relates to a method for producing citric acid.

More particularly, this invention relates to a method for producing citric acid which comprises inoculating a citric acid-accumulating yeast belonging to the genus Candida, capable of utilizing hydrocarbons and incapable of utilizing citric acid in an aqueous culture medium containing, as the main carbon source, at least one normal paraflin containing 9 to 20 carbon atoms in the molecule; incubating the culture at a pH of from about 2 to about 7 until citric acid is substantially accumulated in the culture broth; and recovering the so-accumulated citric acid, therefrom.

Citric acid is in a great demand and used for example, as an acidulant in beverage and in pharmaceutical syrups.

Regarding the production of citric acid by fermentation, processes involving the use of microorganisms such as the molds beyonding to the genera Penicillinm, Aspergillus, etc. are well known and have been the subjects of numerous reports and patents. Those processes, however, invariably depend upon the employment of sugars and other expensive carbon sources, and the fermentation period involved are as long as to 12 days.

On the other hand, it has been disclosed that various yeasts have the ability of accumulating citric acid in a medium containing hydrocarbons.

These yeasts, however, consume gradually the citric acid once produced with the elapse of fermentation time and the final yield depends upon the balance of production and consumption of citric acid.

It is for this reason that the yield of citric acid relative to the carbon sources used has never been as satisfactory as desired.

The present inventors have long engaged in the studies of the metabolism of Candida species and have succeeded in the isolation of certain mutants which lack in the ability to utilize citric acid and are capable of utilizing hydrocarbons to produce and accumulate citric acid in a good yield. The present invention is a result of the above studies.

Production of citric acid by the method of this invention ofiers many advantages.

In the first place, the yield is extremely high. It is believed that this improved yield comes from the suppression of production of other organic acids, mainly, isocitric acid which would otherwise be produced almost to an amount as large as citric acid on consumption of the objective citric acid. The second advantage of this method is in that the fermentation comes to an end in a relatively short time. Thirdly it is extremely easy to determine the timing of harvest and the fermentation can be carried out without worrying about overfermentation, since the citric acid once produced is not consumed by the organism. These are considerable advantages in fermentation control. Furthermore, because of the unusually low concentrations of other organic acids formed as by-products, the isolation of citric acid in a purified form from the culture is greatly facilitated. According to the present invention, the ratio of citric acid to isocitric acid is usually not lower than 9:1.

Thus, the main object of the present invention is to provide a method for producing citric acid in a good yield.

The other object of the present invention is to facilitate the fermentation control and purification procedure of the objective citric acid.

These objects are realized by inoculating a citric acidaccumulating yeast belonging to the genus Candida, capable of utilizing hydrocarbons and incapable of utilizing citric acid in an aqueous culture medium containing, as a main carbon source, at least one normal parafiin containing 9 to 20 carbon atoms in the molecule; incubating the culture at a pH of from about 2 to about 7 until citric acid is substantially accumulated in the culture broth; and recovering the so-accumulated citric acid therefrom.

As the parent strains, from which the aforesaid mutants of the genus Candida can be derived, there may be exemplified such yeasts as Candida lipolytica, Candida parapsilosis, Candida bfumptii, Candida intermedia, Candida tropicalis, Candida guilliermondii, etc., as well as certain yeasts isolated in nature.

Spontaneous or induced mutation is taken advantage of to derive the desired mutants which are incapable of utilizing citric acid and capable of utilizing the abovementioned hydrocarbons as a sole carbon source to produce citric acid. For the mutation, such means may be taken as ultraviolet radiation, C0 treatment, X-ray and other high energy radicals, or chemical treatment with mutagens such as NaNO H 0 N-methyl-N-nitro-N- nitroso-guanidine, acryflavine and the like.

In the present invention, when an organism does not grow any more under such conditions as specified below, the organism is defined as lacking in ability of utilizing citric acid, although there is still a chance of its growing on further incubation.

( l Medium: Percent Trisodiumcitrate 0.05 NH.,NO 0.1 NH,C1 0.1 KH PO 0.1 MgSO '7H O Agar 2.2

Necessary nutrients such as vitamins, amino acids, and nucleic acid constituents, are added thereto in case the microorganisms require such substances for their growth.

(2) Cultural conditions-incubated at 28 C. for 2 days.

Some of those mutants are devoid of the ability to take citric acid into cells and others require certain nutrients such as various vitamins, amino acids, nucleic acid constituents, etc., and even those mutants may be employed for the purposes of this invention, insofar as they lack in the ability to utilize citric acid and retain the ability to utilize hydrocarbons as a sole carbon source to produce citric acid.

Some of the above mentioned mutants and their microbiological features are shown in Table l in comparison with parent strains.

The IFO numbers placed after the yeast employed in the following examples and tables are the respective accession numbers at the Institute for Fermentation, Osaka, Japan.

The various species of the genus Candida utilized in the present invention have been deposited in the American Type Culture Collection in Rockville, Md., U.S.A. The ATCC numbers of the various species contemplated in the instant invention and appearing in the present disclosure and specific examples are as follows:

Candida lipolytica L-36ATCC 20237 4 ing agent, e.g. a surfactant of the type of polyoxyethylene sorbitan monostearate can be employed.

These hydrocarbons are by themselves satisfactory as carbon sources, but, if desired, commonly employable carbon sources such as carbohydrates (e.g. glucose) can be used together with the hydrocarbons.

As the necessary nitrogen sources, use may be made of various inorganic ammonium salts such as NH Cl, (NH SO (NHQ HPO NH OH, NH NO etc., urea, the ammonium salts of various organic acids, eg ammonium acetate, and organic nitrogenous materials such as dried yeast, yeast extract, meat extract, fish meal, soybean fiour, corn steep liquor, peptone, distillers waste and the like. Those nitrogen sources may be used either singly or in combination.

In addition to such carbon and nitrogen sources, the medium to be employed may further contain, if required, any of those inorganic salts which are commonly empioyed, e.g. the salts of iron, manganese, magnesium, cal- Candida sp. H-22-ATCC 20238 cium and various nutrients which might be necessary for Candida sp. FRZ3ATCC 20239 the growth of the mutant.

Candida rropz'calis 9M-ATCC 20240 The pH of the medium may be selected from the wide Candida sp. 164 K1ATCC 20241 range which permits growth of the mutants employed,

TABLE 1 Parent strain number Candida lipolytica Candida tropicalis IFO 1437 Candida sp. Candida s IFO 0589 Candida sp. (ATCC 20114) Il O 1461 lFO 1462 (AICC 20115) 164 IFO 1460 Mutant strain No." Candida lipolyiica Candida sp. Candida sp. Candida tnmimlta Candida sp.

11-36 ATCC 20237 1L2: AlCC 20238 FRZ-3 AlCC 20239 0M ATCC 20240 164 K-1 AICC 20241 Malt extract (1., Circular to ellptical; Circular to ollipitcnl; Circular to elliptical; Circular to elliptical; Circular to elliptical; 3 days. pseudomycelium, pseudomyeelium, budding; pseudobudding; pseudobudding; pseudotruemycelium. myeeliuui. mycelium and myccllum and myoelium and Malt slant, 25 C.,

3 days.

Malt extract, 25 0.,

l7 days 31 days Malt agar, 17 C.,1

Elliptical trueniyceliuni.

Circular to elliptical,

ghostlike, lllyculilllil is fragmented. Dry

with wrinkles formed Elliptical; truemyecliuin, pseutlolnyceliuni.

Oval to elliptical,

truemycelium, no

pellir-le formed; yeast ring.

Yeast ring liable to be detached; no pelllcle.

White to yellow; tl- Y truemyculium. (ircular to elliptical,

truernycelium and pseudcmyuulium. Circular to elliptical,

truvlnycelium, a thin pelllt'le.

Yeast ring. sedimentation islet, pelliclc, cloudiness of medium.

White to yellow; dry

truem ycelium. Circular to elliptical,

truemycelium and pseudomyeeliuni. Circular to elliptical, occasionally thin pelliclc formed, yeast ring.

Yeast ring, no sedimentation, islet,

trucmycelium. Circular to elliptical, truemycelium and pseudomycelium. Circular to elliptical,

no pellicle formed,

growth on the surface.

Thin peliicle formed,

yeast. ring.

Torbid yellow, slightly wrinkled, dry, medium turning brown.

month. and liliny.

llll+ lllll lltl+++ i:

and filmy. yellow, dry, liable. wrinkled, wet.

lilue lue lue.

The medium to be employed in the method of this invention should be slightly modified according to the particular mutants to be used, but such normal alkanes of 9 to 20 carbons in the molecule as n-nonane, n-undecane, n-dodecane, n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane, n-nondaceane, n-eicosane, etc., as well as their mixtures, and such petroleum oil fractions as gas oil, heavy gas oil, kerosene, etc. may be utilized as carbon sources to advantage.

The hydrocarbons are generally used in such an amount of the normal paraifin(s) with 9 to 20 carbon atoms in the molecule as about 3 to 20% (volume/volume) in the culture medium.

As these hydrocarbons are scarcely soluble in water, the addition to an aqueous culture medium is practically carried out under stirring or shaking to prepare a suspension containing very fine particles. If desired, a suspendand generally the range of pH 2 to pH 7 and particularly of pH 3 to pH 6, is preferred. If, in the course of cultivation, a drop in pH occurs by citric acid produced, it is advisable to adjust the pH of the medium from time to time within the afore-said range while continuing the cultivation. For this purpose, the cultivation may be conducted with an occasional addition of a neutralizer which may for example be CaCO Ca(OH) NH,OH, NaOH or Na CO Alternatively, in order to give an adequate buffering action to the medium, CaCO for one, may be preliminarily added in an amount according to the possible pH drop.

The incubation temperature may vary somewhat depending upon the particular mutant employed, and generally be within the range of 20 to 35 C., particularly of 25 to 30 C. Since the method of this invention is generally carried out under aerobic conditions, a stirring cultural method involving the use of a tank is suitable for the purpose. In this connection, it defoaming is required, it is advisable to add such known defoaming agents as polyoxypropylene derivatives, soybean oil, silicone oil, lard oil, etc.

Thus accumulated citric acid in the culture broth is recovered by applying per se conventional means such as filtration, centrifugation, heating, precipitation, crystallization, decolorization and drying, and column-chromatography can be carried out either singly or in combination. It the case demands, the pH of the culture broth is adjusted. By way of illustration, when there exists insoluble calcium citrate in the culture broth, it is desirable to acidify the broth with hydrochloric acid to solubilize the said salt, followed by further separation procedure.

If desired, after being separated from yeast cells, the culture liquid is neutralized with sodium hydroxide, calcium carbonate, milk of lime, or the like. The thus-obtained solution is kept standing at room temperature (about 15 to about 30 C.) or under heating, followed by filtration or centrifugation and drying to give calcium citrate as white powder. The powder is, if required, further purified by per se conventional means such as neutralization, etc.

If the powder is a mixture of citric acid and isocitric acid, the separation may be effected, for instance, by the following means.

The powder dissolved in hot water is subjected to suction-filtration, while hot, to obtain calcium salts of citric acids, followed by neutralization and filtration. The filtrate is concentrated to a syrup state and then subjected to column chromatography on silica gel utilizing an n-butanol-chloroform mixture as an eluting solvent, whereby citric acid and (+)-isocitric acid flow down into different fractions.

Citric acid and (+)-isocitric acid are respectively measured by the pentabromoacetone method (Seikagaku 27, 72 (1955) and by the enzymatic method (Methods of Enzymatic Analysis (Verlag Chemie) 318, Giinther Siebert).

Presently-preferred embodiments of the invention are shown in the following examples, but they are not intended to be construed as a limitation of the present invention. Throughout the specification, percentages are calculated on the weight per volume basis, and yields are calculated on the weight of produced citric acid per weight of consumed normal parafiins.

The relationship between part( by weight and part(s) by volume is the same as that between gram(s) and milliliter(s).

EXAMPLE 1 A culture of Candida lipolytica L-36(ATCC 20237) which has been derived from Candida lipolytica 11 01437 is used to inoculate in parts by volume of a sterilized aqueous medium (pH 6.0) which contains a hydrocarbon mixture composed predominantly of n-paraffin having 13 to 15 carbon atoms (2%), NH Cl(0.2% KH PO (0.05% MgMOy 7H O (0.05%), MnSO -7H O (0.001%), FeSO -7H O (0.001%), and CaCO (0.5%) then incubated at 28 C. under agitation and aeration for 48 hours. 10 parts by volume of the resulting pre-culture is transferred to 100 parts by volume of a medium containing n-parafiin (6%) (same as aboce), NH Cl (0.2%), KH PO, (0.010), MgSO -7H O (0.05%), MnSO -7H O (0.001%), FeSO -7H O (0.001%), dried yeast (0.1%) and CaCO (0.5%), and the inoculated medium is incubated at 28 C. under aeration and agitation. On the second day CaCO (0.5 and on the third day CaCO;, (3%) are aseptically added to adjust the pH of the medium, and the cultivation is continued for a total of 5 days. The yields of citric acid produced and accumulated in the broth and of isocitric acid, which occurs in the highest concentration of all byproduct organic acids, are

shown below, along with the corresponding yields attainable by the cultivation of the parent strain.

A portion of the above culture is further incubated for 3 days, or a total of 8 days, at the end of which time the accumulation of citric acid by the mutant strain has reached 69.1 mg./m1. In contrast, the acumulation by the parent strain rather drops to 13.2 mg./ml.

On the 5th day of cultivation, parts by volume of the culture is taken and adjusted to pH 2.0 by the addition of 3 N HCl, followed by centrifugation to remove the cells. The supernatant fluid is adjusted to pH 6.8 with 5 N NaOH and heated at 100 C. for 30 minutes. After cooling the fluid, the sediment is recovered by filtration and allowed to dry in a stream of air. The procedure yields calcium citrate which contains 6.3 parts by weight of citric acid. The salt is suspended in 40 parts by volume of water and neutralized with 5 N H SO To the supernatant fluid is added 1 part by weight of activated carbon, followed by filtration.

The resulting filtrate is concentrated under reduced pressure until a syrup of low consistency is obtained. (If calcium hydroxide precipitates, they are filtered oil in the course of concentration). The concentrate is allowed to stand in a refrigerator, whereupon 5.7 parts by weight of crystals of citric acid (as citric anhydride) are obtained.

EXAMPLE 2 A citric acid non-utilizing mutant H-22 (ATCC 20238) derived from Candida sp. IFO 1461 (the above mutant has a non-L-lactic acid utilizing property as well) is used to inoculate 100 parts by volume of a sterilized medium (pH 6.5) containing a n-paraffin mixture (8%) having 12 to 18 carbon atoms (purity 92%), NH,Cl (0.3%), KH PO (0.01%), MgSO -7H O (0.05%), FeSO -7H O (0.05%) and CaCO (0.5%). The whole are incubated at 28 C. for 3 days, with NH OH (14% solution) being added from time to time so as to maintain the yellowish green color of the medium (Le. between pH 4 and pH 5). The results are shown below, along with the results attainable by use of the parent organism.

lsocitric acid (the most.

A portion of the above culture is further incubated for 2 days, at the end of which time the yield of citric acid by the mutant is 109 mg./ml. It is worthy of note that the corresponding figure for the parent strain is as low as 25.4 mg./ml.

On the third day, the above culture is collected and filtered to remove the organisms. The filtrate is adjusted to pH 7.0 by the addition of Ca(OH) and boiled under slightly reduced pressure for 30 minutes. After cooling, the sediment is recovered by filtration and treated in the same manner as Example 1. The procedure yields 9.3 parts by weight of (as citric anhydride) crystals of citric acid.

EXAMPLE 3 Candida sp. FRZ-3 (ATCC 20239), derived from Candida sp. IFO 1462, is cultivated in the same manner as in Example 2, and on the third day of cultivation, the

culture is analyzed for citric acid and byproduct isocitric acid. The results are shown below.

Candida sp. Kl (ATCC 20241) (derived from Candida sp. 164 IFO 1460) and Candida tropicalis 9M (ATCC 20240) (derived from Candida tropicalis IFO 0589) are cultivated in the same manner as in Example 2 and on the third day of cultivation, the cultures are analyzed for citric acid and isocitric acid.

The results are shown below in comparison with those of their parents.

Citric acid Isocitric acid Percent yield Percent yield Yield, relative to Yield, relative to rug/ml. carbon source rug/n11. carbon source Candida sp. K l

ATCC 20241 81 100.1 9 11.2 Candida sp. 1G4

(lFL) llti) 32 30 16 20 Candida tropicalis 9M ATCC 20240.. 88 110 2 2. 5 Candida tropicalis (IFO 0589) 46 57. 5 32 40 What we claim is:

1. A method for producing citric acid which comprises inoculating a citric acid-accumulating mutant incapable of utilizing citric acid belonging to the genus Candida selected from the group consisting of Candida lipolytica L-37 ATCC-20237 Candida sp. H22 ATCC NO. 24238 Candida sp. F RZ-3 ATCC No. 20239 Candida tropicalis 9M ATCC 20240 Candida sp. 164 K-l ATCC No. 20241 in an aqueous culture medium containing, as the main carbon source, at least one normal parafiin containing 9 to 20 carbon atoms in the molecule; incubating the culture at a pH of from about 2 to about 7 until citric acid is substantially accumulated in the culture broth and recovering so-accumulated citric acid therefrom.

2. A method according to claim 1, wherein the incubation temperature is between 20 and C.

3. A method according to claim 1, wherein the pH of culture medium is kept between 3 and 6.

4. A method according to claim 1, wherein the yeast is Candida lipolytica L-37 ATCC 20237.

5. A method according to claim 1, wherein the yeast is Candida sp. H22 ATCC No. 20238.

6. A method according to claim 1, wherein the yeast is Candida sp. FRZ-3 ATCC No. 20239.

7. A method according to claim 1, wherein the yeast is Candida tropicalis 9M ATCC 20240.

8. A method according to claim 1, wherein the yeast is Candida sp. 164 K-l ATCC No. 20241.

References Cited FOREIGN PATENTS 716,247 11/1968 Belgium.

A. LOUIS MONACELL, Primary Examiner G. M. NATH, Assistant Examiner 

